Anti-MARCO antibody [RM2074]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MARCO antibody [RM2074] (AB322705)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labelling MARCO with ab322705 at 1/100 (5.16 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing increased cytoplasmic staining in human PBMC (shown in green) treated with M-CSF (500 ng/mL) for 72 hr and then IL-10 (20 ng/mL) for 24 hr. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• Flow Cyt

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Flow Cytometry - Anti-MARCO antibody [RM2074] (AB322705)

Flow cytometric analysis of J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage, upper left and right) untreated / J774A.1 cells treated with 1ug/mL LPS for 24 hours (lower left and right) cells labelling MARCO with ab322705 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 488, ab150077) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

• Flow Cyt

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Flow Cytometry - Anti-MARCO antibody [RM2074] (AB322705)

Flow cytometric analysis of Human PBMC (upper left and right) / Human PBMC treated with 50ng/mL M-CSF for 72 hours and 20ng/mL IL-10 for 24 hours (lower left and right) cells labelling MARCO with ab322705 at 1/500 dilution (0.1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 488, ab150077) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with anti human CD14 conjugated to BV421.
Gated on viable Monocytes.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MARCO antibody [RM2074] (AB322705)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) cells labelling MARCO with ab322705 at 1/50 (10.32 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing increased cytoplasmic staining in J774A.1 cells (shown in green) treated with lipopolysaccharide (1 ug/mL) for 24 hr. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-MARCO antibody [RM2074] (AB322705)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh frozen) tissue labeling MARCO with ab322705 at 1/500 (1.032 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Confocal image showing positive staining in red pulp of mouse spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with ab322705 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-MARCO antibody [RM2074] (AB322705)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat liver (fresh frozen) tissue labeling MARCO with ab322705 at 1/500 (1.032 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Confocal image showing positive staining in Kupffer cells of rat liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab322705 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MARCO antibody [RM2074] (AB322705)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling MARCO with ab322705 at 1/500 dilution.

Positive staining on mouse spleen

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-MARCO antibody [RM2074] (AB322705)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse pancreas (fresh frozen) tissue labeling MARCO with ab322705 at 1/500 (1.032 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Negative control : confocal image showing no staining on mouse pancreas. The nuclear counterstain was DAPI (Blue). The section was incubated with ab322705 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-MARCO antibody [RM2074] (AB322705)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat pancreas (fresh frozen) tissue labeling MARCO with ab322705 at 1/500 (1.032 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Negative control : confocal image showing no staining on rat pancreas. The nuclear counterstain was DAPI (Blue). The section was incubated with ab322705 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-MARCO antibody [RM2074] (AB322705)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (fresh frozen) tissue labeling MARCO with ab322705 at 1/500 (1.032 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Confocal image showing positive staining in Kupffer cells of mouse liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab322705 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MARCO antibody [RM2074] (AB322705)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling MARCO with ab322705 at 1/500 dilution.

Positive staining on mouse liver

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

Supplier Data

Immunoprecipitation - Anti-MARCO antibody [RM2074] (AB322705)

MARCO was immunoprecipitated from 0.35 mg Mouse lymph node tissue lysate with ab322705 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322705 at 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse lymph node tissue lysate
Lane 2 : ab322705 IP in Mouse lymph node tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322705 in mouse lymph node tissue lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-MARCO antibody [RM2074] (ab322705) at 1/30 dilution

All lanes:

Mouse lymph node tissue lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 8s

• IP

Supplier Data

Immunoprecipitation - Anti-MARCO antibody [RM2074] (AB322705)

MARCO was immunoprecipitated from 0.35 mg Rat liver tissue lysate with ab322705 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322705 at 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Rat liver tissue lysate
Lane 2 : ab322705 IP in Rat liver tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322705 in rat liver tissue lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-MARCO antibody [RM2074] (ab322705) at 1/30 dilution

All lanes:

Rat liver tissue lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-MARCO antibody [RM2074] (AB322705)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : pancreas, brain.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 26892079).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lanes 1-3, 8-9 : 180 seconds, lanes 4-7 : 37 seconds

All lanes:

Western blot - Anti-MARCO antibody [RM2074] (ab322705) at 1/1000 dilution

Lane 1:

Human lung tissue lysate at 20 µg

Lane 2:

Human pancreas tissue lysate at 20 µg

Lane 3:

Mouse spleen tissue lysate at 20 µg

Lane 4:

Mouse lymph node tissue lysate at 20 µg

Lane 5:

Mouse pancreas tissue lysate at 20 µg

Lane 6:

Rat lymph node tissue lysate at 20 µg

Lane 7:

Rat pancreas tissue lysate at 20 µg

Lane 8:

Rat liver tissue lysate at 20 µg

Lane 9:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60-75 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-MARCO antibody [RM2074] (AB322705)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 26892079).

The expression of MARCO/Macrophage receptor is induced by Lipopolysaccharides (LPS) treatment (PMID : 9916718).

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-MARCO antibody [RM2074] (ab322705) at 1/1000 dilution

Lane 1:

Untreated J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) whole cell lysate at 20 µg

Lane 2:

J774A.1 treated with 1 ug/ml LPS for 24 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60-75 kDa,36 kDa

true

Exposure time: 103s